Antibody and uses thereof

ABSTRACT

Described are specific binding members e.g. antibodies which may be used in the treatment of diseases associated with cathepsin S activity. The specific binding members bind cathepsin S and inhibit its proteolytic activity. The binding members may be used in the treatment of diseases such as cancer, inflammatory diseases, neurodegenerative disorders, autoimmune disorders, and other diseases associated with excessive, deregulated or inappropriate angiogenesis.

FIELD OF THE INVENTION

This application relates to a specific binding member and its use inmethods of treatment. In particular, it relates to specific bindingmembers to cathepsin S, preferably specific binding members whichinhibit the proteolytic activity of cathepsin S.

BACKGROUND TO THE INVENTION

Proteases are a large group of proteins that comprise approximately 2%of all gene products (Rawlings and Barrett, 1999). Proteases catalysethe hydrolysis of peptide bonds and are vital for the proper functioningof all cells and organisms. Proteolytic processing events are importantin a wide range of cellular processes including bone formation, woundhealing, angiogenesis and apoptosis.

The lysosomal cysteine proteases were initially thought to be enzymesthat were responsible for non-selective degradation of proteins in thelysosomes. They are now known to be accountable for a number ofimportant cellular processes, having roles in apoptosis, antigenpresentation, coagulation, digestion, pro-hormone processing andextracellular matrix remodelling (Chapman et al, 1997).

Cathepsin S (Cat S) is a member of the papain superfamily of lysosomalcysteine proteases. To date, eleven human cathepsins have beenidentified, but the specific in vivo roles of each are still to bedetermined (Katunuma et al, 2003). Cathepsins B, L, H, F, O, X and C areexpressed in most cells, suggesting a possible role in regulatingprotein turnover, whereas Cathepsins S, K, W and V are restricted toparticular cells and tissues, indicating that they may have morespecific roles (Kos et al, 2001; Berdowska, 2004).

Cat S was originally identified from bovine lymph nodes and spleen andthe human form cloned from a human macrophage cDNA library (Shi et al,1992). The gene encoding Cat S is located on human chromosome 1q21. The996 base pair transcript encoded by the Cat S gene, is initiallytranslated into an unprocessed precursor protein with a molecular weightof 37.5 kDa. The unprocessed protein is composed of 331 amino acids; a15 amino acid signal peptide, a 99 amino acid pro-peptide sequence and a217 amino acid peptide. Cat S is initially expressed with a signalpeptide that is removed after it enters the lumen of the endoplasmicreticulum. The propeptide sequence binds to the active site of theprotease, rendering it inactive until it has been transported to theacidic endosomal compartments, after which the propeptide sequence isremoved and the protease is activated (Baker et al, 2003).

Cat S has been identified as a key enzyme in major histocompatibilitycomplex class II (MHC-II) mediated antigen presentation, by cleavage ofthe invariant chain, prior to antigen loading. Studies have shown thatmice deficient in Cat S have an impaired ability to present exogenousproteins by APC's (Nakagawa et al, 1999). The specificity of Cat S inthe processing of the invariant chain Ii, allows for Cat S specifictherapeutic targets in the treatment of conditions such as asthma andautoimmune disorders (Chapman et al, 1997).

Pathological Association of Cat S

Alterations in protease control frequently underlie many humanpathological processes. The deregulated expression and activity of thelysosomal cysteine protease Cathepsin S has been linked to a range ofconditions including neurodegenerative disorders, autoimmune diseasesand certain malignancies.

Cat S upregulation has been linked to several neurodegenerativedisorders. It is believed to have a role in the production of the βpeptide (Aβ) from the amyloid precursor protein (APP) (Munger et al,1995) and its expression has been shown to be upregulated in bothAlzheimer's Disease and Down's Syndrome (Lernere et al, 1995). Cat S mayalso have a role in Multiple Sclerosis through the ability of Cat S todegrade myelin basic protein, a potential autoantigen implicated in thepathogenesis of MS (Beck et al, 2001) and in Creutzfeldt-Jakob disease(CJD) patients, Cat S expression has been shown to increase more thanfour fold (Baker et al, 2002).

Aberrant Cat S expression has also been associated with atherosclerosis.Cat S expression is negligible in normal arteries, yet human atheromadisplay strong immunoreactivity (Sukhova et al, 1998). Further studiesusing knockout mice, deficient in both Cat S and the LDL-receptor, wereshown to develop significantly less atherosclerosis (Sukhova et al,2003). Further research has linked Cat S expression with inflammatorymuscle disease and rheumatoid arthritis. Muscle biopsy specimens frompatients with inflammatory myopathy had a 10 fold increase in Cat Sexpression compared to control muscle sections (Wiendl et al, 2003), andlevels of Cat S expression were significantly higher in synovial fluidfrom patients with rheumatoid arthritis compared to those withosteoarthritis (Hashimoto et al, 2001).

The role of Cat S has also been investigated in specific malignancies.The expression of Cat S was shown to be significantly greater in lungtumour and prostate carcinomas sections in comparison to normal tissue(Kos et al, 2001, Fernandez et al, 2001) and suggested that Cat S mayhave a role in tumour invasion and disease progression.

Recent work in this laboratory on Cat S demonstrated the significance ofits expression in human astrocytomas (Flannery et al, 2003).Immunohistochemical analysis showed the expression of Cat S in a panelof astrocytoma biopsy specimens from WHO grades I to IV, but appearedabsent from normal astrocytes, neurones, oligodendrocytes andendothelial cells. Cat S expression appeared highest in grade IV tumoursand levels of extracellular activity were greatest in cultures derivedfrom grade IV tumours.

Cat S has been shown to be active in the degradation of ECMmacromolecules such as laminin, collagens, elastin and chondroitinsulphate proteoglycans (Liuzzo et al, 1999).

The generation of inhibitors specifically targeting Cat S have potentialas therapeutic agents for alleviations of the symptoms associated withthe activity of this protease.

Inhibition of Cat S

When proteases are over-expressed, therapeutic strategies have focusedon the development of inhibitors to block the activity of these enzymes.The generation of specific small molecule inhibitors to the cathepsinshave proved difficult in the past, due to problems with selectivity andspecificity. The dipeptide α-keto-α-aldehydes developed as potentreversible inhibitors to Cat S by Walker et al, had the ability toinhibit Cat B and L, albeit with less efficiency (Walker et al, 2000),and the Cat S inhibitor 4-Morpholineurea-Leu-HomoPhe-vinylsulphone(LHVS) has also been shown to inhibit other cathepsins when used athigher concentrations (Palmer et al, 1995).

SUMMARY OF THE INVENTION

As described herein, the present inventors have developed a monoclonalantibody with specificity for cathepsin S which potently inhibits theproteolytic activity of cathepsin S, and moreover, inhibits tumour cellinvasion and angiogenesis. The inventors have identified the VH and VLdomains and CDRs of the antibody. This is the first demonstration of acathepsin S specific antibody directly inhibiting the protease activityof cathepsin S and thus uniquely enables the use of such antibodies asactive therapeutic agents with a wide range of applications from cancertherapeutics to anti-inflammatory agents with high specificity and lowtoxicity.

Accordingly, in a first aspect, the present invention provides aspecific binding member which binds cathepsin S and inhibits itsproteolytic activity.

In one embodiment, the specific binding member comprises an antigenbinding domain comprising at least one of the CDRs with an amino acidsequence selected from the group consisting of Seq ID No: 1, Seq ID No:2, Seq ID No: 3, or a variant thereof and/or at least one of the CDRswith an amino acid sequence consisting of Seq ID No: 4, Seq ID No: 5 andSeq ID No: 6, or a variant thereof.

The amino acid sequences corresponding to SEQ ID NOS: 1-6 are asfollows: Seq ID No: 1: SYDMS Seq ID No: 2: YITTGGVNTYYPDTVKG Seq ID No:3 HSYFDY Seq ID No: 4: RSSQSLVHSNGNTYLH Seq ID No: 5: KVSNRFS Seq ID No:6: SQTTHVPPT

In one embodiment of the first aspect of the invention, the specificbinding member comprises an antigen binding domain comprising at leastone of the CDRs with an amino acid sequence selected from the groupconsisting of Seq ID No: 1, Seq ID No: 2, Seq ID No: 3, and/or at leastone of the CDRs with an amino acid sequence consisting of Seq ID No: 4,Seq ID No: 5 and Seq ID No: 6.

In an embodiment, the specific binding member comprises an antigenbinding domain comprising at least one, for example at least two or allthree of the CDRs with an amino acid sequence selected from the groupconsisting of Seq ID No: 1, Seq ID No: 2, Seq ID No: 3, or variantsthereof and at least one, for example at least two, for example allthree of the CDRs with an amino acid sequence consisting of Seq ID No:4, Seq ID No: 5 and Seq ID No: 6, or variants thereof.

In a particular embodiment, the specific binding member comprises a CDRhaving the amino acid sequence SEQ ID NO: 5, or a variant thereof and/orthe CDR having the amino acid sequence SEQ ID NO: 6, or a variantthereof.

In one embodiment, the specific binding member comprises an antibodyV_(H) domain or an antibody V_(L) domain, or both.

In one embodiment, the specific binding the antibody V_(H) domaincomprises at least one of the CDRS, for example two or three CDRS, withan amino acid sequence selected from the group consisting of Seq ID No:1, Seq ID No: 2, Seq ID No: 3, and/or the antibody V_(L) domaincomprises at least one of the CDRS, for example two or three CDRS, withan amino acid sequence consisting of Seq ID No: 4, Seq ID No: 5 and SeqID No: 6.

In a preferred embodiment, the antibody V_(L) domain comprises the aminoacid sequence Seq ID No: 8 and/or the antibody V_(H) domain comprisesthe amino acid sequence Seq ID No: 7. Seq ID No: 7:VQLQESGGVLVKPGGSLKLSCAASGFAFSSYDMSWVRQTPEKRLEWVAYITTGGVNTYYPDTVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCARHSY FDYWGQGTTVTVSS Seq IDNo: 8: DVLMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQTTHVP PTFGSGTKLEIKR

The specific binding member may be an antibody, for example a wholeantibody.

In one alternative embodiment, the specific binding member may be anantibody fragment such as an scFv.

The provision of the specific binding members of the present inventionenables the development of related antibodies which also inhibit theproteolytic activity of cathepsin S and which optionally have similar orgreater binding specificity.

Accordingly, further encompassed within the scope of the first aspect ofthe present invention are specific binding members comprising at leastone of the CDRs with an amino acid sequence selected from the groupconsisting of Seq ID No: 1, Seq ID No: 2, Seq ID No: 3, and/or at leastone of the CDRs with an amino acid sequence consisting of Seq ID No: 4,Seq ID No: 5 and Seq ID No: 6, in which 5 or less, for example 4, 3, 2,or 1 amino acid substitutions have been made in at least one CDR andwherein the specific binding member retains the ability to inhibit theproteolytic activity of cathepsin S.

In an embodiment of the first aspect of the invention, the specificbinding member of the first aspect of the invention has the ability toinhibit tumour cell invasion.

In another embodiment, the specific binding member of the first aspectof the invention has the ability to inhibit angiogenesis.

In a second aspect of the invention, there is provided a nucleic acidencoding a specific binding member according to the first aspect of theinvention.

The nucleic acid may be used to provide specific binding membersaccording to the first aspect of the invention. Accordingly, there isprovided a method of producing a specific binding member capable ofinhibiting the proteolytic activity of cathepsin S, said methodcomprising expressing the nucleic acid according to the second aspect ofthe invention in a host cell and isolating said specific binding memberfrom said cell.

A further aspect of the invention is a pharmaceutical compositioncomprising a specific binding member of the first aspect of theinvention or a nucleic acid of the second aspect of the invention.

The specific binding members, nucleic acids or compositions of theinvention may be used for the inhibition of cathepsin S activity, inparticular where the cathepsin S is aberrantly active. Typically, innon-disease states, cathepsin S is localised intracellularly, withinlysosomes. However, in certain disease states, cathepsin S is secretedfrom cells.

Accordingly, in a further aspect, the present invention provides amethod of inhibiting cathepsin S in a biological sample, said methodcomprising administration of a specific binding member according to thefirst aspect of the present invention or a nucleic acid according to thesecond aspect of the invention.

In a further aspect, there is provided a method of treating a conditionassociated with activity of cathepsin S in a patient in need oftreatment thereof, said method comprising administration of a specificbinding member according to the first aspect of the present invention ora nucleic acid according to the second aspect of the invention.

In one embodiment, condition is a condition associated with aberrantactivity of cathepsin S.

In the context of the present application, cathepsin S is considered tobe aberrantly active where its expression or localisation differs fromthat of normal healthy cells, for example overexpression and/orsecretion from a cell which, typically, does not secrete cathepsin S,extracellular localisation, cell surface localisation, in whichcathepsin S is not normally expressed at the cell surface, or secretionor expression which is greater than normal at or from a cell or tissueand where its activity contributes to a disease state.

Further provided is a specific binding member according to the firstaspect of the invention or a nucleic acid according to the second aspectof the invention for use in medicine.

The invention further provides a specific binding member according tothe first aspect of the invention or a nucleic acid according to thesecond aspect of the invention for use in treatment of a conditionassociated with aberrant cathepsin S activity

Also provided is the use of a specific binding member according to thefirst aspect of the invention or a nucleic acid according to the secondaspect of the invention in the preparation of a medicament for thetreatment of a condition associated with aberrant activity expression ofcathepsin S.

The invention may be used in the treatment of any condition with whichaberrant activity of cathepsin S is associated, in particular conditionsassociated with expression of cathepsin S. For example, conditions inwhich the invention may be used include, but are not limited toneurodegenerative disorders, for example Alzheimer's disease andmultiple sclerosis, autoimmune disorders, inflammatory disorders, forexample inflammatory muscle disease, rheumatoid arthritis and asthma,atherosclerosis, neoplastic disease, and other diseases associated withexcessive, deregulated or inappropriate angiogenesis.

DETAILED DESCRIPTION Binding Members

In the context of the present invention, a “binding member” is amolecule which has binding specificity for another molecule, themolecules constituting a pair of specific binding members. One member ofthe pair of molecules may have an area which specifically binds to or iscomplementary to a part or all of the other member of the pair ofmolecules. The invention is particularly concerned with antigen-antibodytype reactions.

In the context of the present invention, an “antibody” should beunderstood to refer to an immunoglobulin or part thereof or anypolypeptide comprising a binding domain which is, or is homologous to,an antibody binding domain. Antibodies include but are not limited topolyclonal, monoclonal, monospecific, polyspecific antibodies andfragments thereof and chimeric antibodies comprising an immunoglobulinbinding domain fused to another polypeptide.

Intact (whole) antibodies comprise an immunoglobulin molecule consistingof heavy chains and light chains, each of which carries a variableregion designated VH and VL, respectively. The variable region consistsof three complementarity determining regions (CDRs, also known ashypervariable regions) and four framework regions (FR) or scaffolds. TheCDR forms a complementary steric structure with the antigen molecule anddetermines the specificity of the antibody.

Fragments of antibodies may retain the binding ability of the intactantibody and may be used in place of the intact antibody. Accordingly,for the purposes of the present invention, unless the context demandsotherwise, the term “antibodies” should be understood to encompassantibody fragments. Examples of antibody fragments include Fab, Fab, F(ab′)₂, Fd, dAb, and Fv fragments, scFvs, bispecific scFvs, diabodies,linear antibodies (see U.S. Pat. No. 5,641,870, Example 2 Zapata et al.,Protein Eng 8 (10): 1057-1062 [1995]); single-chain antibody molecules;and multispecific antibodies formed from antibody fragments.

The Fab fragment consists of an entire L chain (VL and CL), togetherwith VH and CH1. Fab′ fragments differ from Fab fragments by havingadditional few residues at the carboxy terminus of the CH1 domainincluding one or more cysteines from the antibody hinge region. The F(ab′) 2 fragment comprises two disulfide linked Fab fragments.

Fd fragments consist of the VH and CH1 domains.

Fv fragments consist of the VL and VH domains of a single antibody.

Single-chain Fv fragments are antibody fragments that comprise the VHand VL domains connected by a linker which enables the scFv to form anantigen binding site. (see Pluckthun in The Pharmacology of MonoclonalAntibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, NewYork, pp. 269-315 (1994).

Diabodies are small antibody fragments prepared by constructing scFvfragments (see preceding paragraph) with short linkers (about 5-10residues) between the VH and VL domains such that inter-chain but notintra-chain pairing of the V domains is achieved, resulting in amultivalent fragment, i.e. a fragment having two antigen-binding sites(see, for example, EP 404 097; WO 93/11161; and Hollinger et al., Proc.Natl. Acad. Sci. USA, 90: 6444-6448 (1993))

Further encompassed by fragments are individual CDRS.

In the present invention, the amino acid sequences of the VH and VLregions of the intact antibody 1E11 with specificity for Cat S has beenidentified. Furthermore, the inventors have identified the six CDRs ofthis antibody (Seq ID Nos: 1, 2, 3, 4, 5 and 6).

As described above, the specific binding members of the presentinvention is not limited to the specific sequences of the 1E11 antibody,the VH, VL and the CDRs having the sequences disclosed herein but alsoextends to variants thereof which maintain the ability to inhibit theproteolytic activity of Cat S. Thus, the CDR amino acid sequences inwhich one or more amino acid residues are modified may also be used asthe CDR sequence. The modified amino acid residues in the amino acidsequences of the CDR variant are preferably 30% or less, more preferably20% or less, most preferably 10% or less, within the entire CDR. Suchvariants may be provided using the teaching of the present applicationand techniques known in the art. The CDRs may be carried in a frameworkstructure comprising an antibody heavy or light chain sequence or partthereof. Preferably such CDRs are positioned in a location correspondingto the position of the CDR(s) of naturally occurring VH and VL domains.The positions of such CDRs may be determined as described in Kabat etal, Sequences of Proteins of Immunological Interest, US Dept of Healthand Human Services, Public Health Service, Nat'l Inst. of Health, NIHPublication No. 91-3242, 1991 and online at www.kabatdatabase.comhttp://immuno.bme.nwu.edu.

Furthermore, modifications may alternatively or additionally be made tothe Framework Regions of the variable regions. Such changes in theframework regions may improve stability and reduce immunogenicity of theantibody.

The antibodies of the invention herein include “chimeric” antibodies inwhich a portion of the heavy and/or light chain is identical with orhomologous to corresponding sequences in antibodies derived from aparticular species or belonging to a particular antibody class orsubclass, while. the remainder of the chain (s) is identical with orhomologous to corresponding sequences in antibodies derived from anotherspecies or belonging to another antibody class or subclass, as well asfragments of such antibodies, so long as they exhibit the desiredbiological activity (see U.S. Pat. No. 4,816,567; and Morrison et al.,Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984)). Chimeric antibodiesof interest herein include “primatized” antibodies comprising variabledomain antigen-binding sequences derived from a non-human primate (e.g.Old World Monkey, Ape etc), and human constant region sequences.

Production Of Specific Binding Members

Specific binding members of and for use in the present invention may beproduced in any suitable way, either naturally or synthetically. Suchmethods may include, for example, traditional hybridoma techniques(Kohler and Milstein (1975) Nature, 256:495-499), recombinant DNAtechniques (see e.g. U.S. Pat. No. 4,816,567), or phage displaytechniques using antibody libraries (see e.g. Clackson et al. (1991)Nature, 352: 624-628 and Marks et al. (1992) Bio/Technology, 10:779-783). Other antibody production techniques are described inAntibodies: A Laboratory Manual, eds. Harlow et al., Cold Spring HarborLaboratory, 1988.

Traditional hybridoma techniques typically involve the immunisation of amouse or other animal with an antigen in order to elicit production oflymphocytes capable of binding the antigen. The lymphocytes are isolatedand fused with a myeloma cell line to form hybridoma cells which arethen cultured in conditions which inhibit the growth of the parentalmyeloma cells but allow growth of the antibody producing cells. Thehybridoma may be subject to genetic mutation, which may or may not alterthe binding specificity of antibodies produced. Synthetic antibodies canbe made using techniques known in the art (see, for example, Knappik etal, J. Mol. Biol. (2000) 296, 57-86 and Krebs et al, J. Immunol. Meth.(2001) 2154 67-84.

Modifications may be made in the VH, VL or CDRs of the binding members,or indeed in the FRs using any suitable technique known in the art. Forexample, variable VH and/or VL domains may be produced by introducing aCDR, e.g. CDR3 into a VH or VL domain lacking such a CDR. Marks et al.(1992) Bio/Technology, 10: 779-783 describe a shuffling technique inwhich a repertoire of VH variable domains lacking CDR3 is generated andis then combined with a CDR3 of a particular antibody to produce novelVH regions. Using analogous techniques, novel VH and VL domainscomprising CDR derived sequences of the present invention may beproduced.

Accordingly, in one embodiment of the invention, the present inventionprovides a method of generating a specific binding member havingspecificity for Cat S, the method comprising: (a) providing a startingrepertoire of nucleic acids encoding a variable domain, wherein thevariable domain includes a CDR1, CDR2 or CDR3 to be replaced or thenucleic acid lacks an encoding region for such a CDR; (b) combining therepertoire with a donor nucleic acid encoding an amino acid sequencehaving the sequence as shown as Seq ID No: 1, 2, 3, 4, 5 or 6 hereinsuch that the donor nucleic acid is inserted into the CDR region in therepertoire so as to provide a product repertoire of nucleic acidsencoding a variable domain; (c) expressing the nucleic acids of theproduct repertoire; (d) selecting a specific antigen-binding fragmentspecific for CatS; and (e) recovering the specific antigen-bindingfragment or nucleic acid encoding it. The method may include an optionalstep of testing the specific binding member for ability to inhibit theproteolytic activity of cathepsin S.

Alternative techniques of producing variant antibodies of the inventionmay involve random mutagenesis of gene(s) encoding the VH or VL domainusing, for example, error prone PCR (see Gram et al, 1992, P.N.A.S. 893576-3580. Additionally or alternatively, CDRs may be targeted formutagenesis e.g. using the molecular evolution approaches described byBarbas et al 1991 PNAS 3809-3813 and Scier 1996 J Mol Biol 263 551-567.

Having produced such variants, antibodies and fragments may be testedfor binding to Cat S and for the ability to inhibit the proteolyticactivity of cathepsin S.

As described herein, the inventors have demonstrated that specificbinding members according to the invention have an anti-proteolyticeffect. Furthermore anti invasive and anti-angiogenic activity has beendemonstrated, as described in the Examples. This therefore enables theuse of the specific binding members of the invention as activetherapeutic agents. Accordingly in one embodiment of the invention, thespecific binding member is a “naked,” specific binding member. A “naked”specific binding member is a specific binding member which is notconjugated with an “active therapeutic agent”.

In the context of the present application, an “active therapeutic agent”is a molecule or atom which is conjugated to a antibody moiety(including antibody fragments, CDRs etc) to produce a conjugate.Examples of such “active therapeutic agents” include drugs, toxins,radioisotopes, immunomodulators, chelators, boron compounds, dyes,nanoparticles etc.

In another embodiment of the invention, the specific binding member isin the form of an immunoconjugate, comprising an antibody fragmentconjugated to an “active therapeutic agent”.

Methods of producing immunoconjugates are well known in the art; forexample, see U.S. Pat. No. 5,057,313, Shih et al., Int. J. Cancer 41:832-839 (1988); Shih et al., Int. J. Cancer 46: 1101-1106 (1990), Wong,Chemistry Of Protein Conjugation And Cross-Linking (CRC Press 1991);Upeslacis et al., “Modification of Antibodies by Chemical Methods,” inMonoclonal Antibodies: Principles And Applications, Birch et al. (eds.),pages 187-230 (Wiley-Liss, Inc. 1995); Price, “Production andCharacterization of Synthetic Peptide-Derived Antibodies,” in MonoclonalAntibodies: Production, Engineering And Clinical Application, Ritter etal. (eds.), pages 60-84 (Cambridge University Press 1995).

The specific binding members of the invention may comprise furthermodifications. For example the antibodies can be glycosylated,pegylated, or linked to albumin or a nonproteinaceous polymer. Thespecific binding member may be in the form of an immunoconjugate.

Antibodies of the invention may be labelled. Labels which may be usedinclude radiolabels, enzyme labels such as horseradish peroxidase,alkaline phosphatase, or biotin.

The ability of a specific binding member to inhibit the proteolyticactivity of cathepsin S may be tested using any suitable method. Forexample the ability of a specific binding member to inhibit theproteolytic activity of cathepsin S may be tested using a fluorimetricassay as detailed in the Examples. In such an assay, any suitablefluorigenic substrate may be used, for example Cbz-Val-Val-Arg-AMC asused in the Examples. A specific binding member is considered to inhibitthe proteolytic activity of cathepsin S if it has the ability to inhibitits activity by a statistically significant amount. For example, in oneembodiment, the specific binding member is able to inhibit theinhibitory activity by at least 10%, for example at least 10%, at least20%, at least 30%, at least at least 40%, at least 50%, at least 60%, atleast 70%, at least 80% or at least 90% when compared to an appropriatecontrol antibody.

The ability of a specific binding member to inhibit tumour cell invasionmay be tested using any suitable invasion assay known in the art. Forexample, such ability may be tested using a modified Boyden chamber asdescribed in the Examples. The specific binding member may be testedusing any suitable tumour cell line, for example a prostate carcinomacell line, e.g. PC3, an astrocytoma cell line e.g. U251 mg, a colorectalcarcinoma cell line, e.g. HCT116, or a breast cancer cell line, e.g.MDA-MB-231 or MCF7. A specific binding member is considered to inhibittumour cell invasion if it has the ability to inhibit invasion by astatistically significant amount. For example, in one embodiment, thespecific binding member is able to inhibit invasion by at least 10%, forexample at least 25%, at least 50%, at least 60%, at least 70%, at least80% or at least 90% when compared to an appropriate control antibody.

The ability of a specific binding member to inhibit angiogenesis may betested using any suitable assay known in the art. For example, suchability may be tested using a Matrigel based assay as described in theExamples.

In one embodiment of the invention, a specific binding member for use inthe invention may have inhibitory activity (for example to inhibit theproteolytic activity or invasion) with a potency of at least 25%, forexample at least 40%, for example at least 50% of the inhibitory potencyof an antibody comprising an antibody V_(H) domain having the amino acidsequence Seq ID No: 7 and an antibody V_(L) domain having the amino acidsequence Seq ID No: 8, when each is compared at its own IC₅₀.

Nucleic Acid

Nucleic acid of and for use in the present invention may comprise DNA orRNA. It may be produced recombinantly, synthetically, or by any meansavailable to those in the art, including cloning using standardtechniques.

The nucleic acid may be inserted into any appropriate vector. A vectorcomprising a nucleic acid of the invention forms a further aspect of thepresent invention. In one embodiment the vector is an expression vectorand the nucleic acid is operably linked to a control sequence which iscapable of providing expression of the nucleic acid in a host cell. Avariety of vectors may be used. For example, suitable vectors mayinclude viruses (e.g. vaccinia virus, adenovirus, etc.), baculovirus);yeast vectors, phage, chromosomes, artificial chromosomes, plasmids, orcosmid DNA.

The vectors may be used to introduce the nucleic acids of the inventioninto a host cell. A wide variety of host cells may be used forexpression of the nucleic acid of the invention. Suitable host cells foruse in the invention may be prokaryotic or eukaryotic. They includebacteria, e.g. E. coli, yeast, insect cells and mammalian cells.Mammalian cell lines which may be used include Chinese hamster ovarycells, baby hamster kidney cells, NSO mouse melanoma cells, monkey andhuman cell lines and derivatives thereof and many others.

A host cell strain that modulates the expression of, modifies, and/orspecifically processes the gene product may be used. Such processing mayinvolve glycosylation, ubiquitination, disulfide bond formation andgeneral post-translational modification.

Accordingly, the present invention also provides a host cell, whichcomprises one or more nucleic acid or vectors of the invention.

Also encompassed by the invention is a method of production of aspecific binding member of the invention, the method comprisingculturing a host cell comprising a nucleic acid of the invention underconditions in which expression of the nucleic specific binding membersfrom the nucleic acid occurs and, optionally, isolating and/or purifyingthe specific binding member.

For further details relating to known techniques and protocols formanipulation of nucleic acid, for example, in preparation of nucleicacid constructs, mutagenesis, sequencing, introduction of DNA into cellsand gene expression, and analysis of proteins, see, for example, CurrentProtocols in Molecular Biology, 5th ed., Ausubel et al. eds., John Wiley& Sons, 2005 and, Molecular Cloning: a Laboratory Manual: 3^(rd) editionSambrook et al., Cold Spring Harbor Laboratory Press, 2001.

Treatment

The specific binding members and nucleic acids of the invention may beused in the treatment of a number of medical conditions.

Treatment” includes any regime that can benefit a human or non-humananimal. The treatment may be in respect of an existing condition or maybe prophylactic (preventative treatment). Treatment may includecurative, alleviation or prophylactic effects.

The specific binding members and nucleic acids of the invention may beused in the treatment of a variety of condition and disorders. Theseinclude atherosclerosis and neoplastic disease, neurodegenerativedisorders, autoimmune diseases, cancer, inflammatory disorders, asthma,and atherosclerosis, and pain.

Neurodegenerative disorders which may be treated using the bindingmembers, nucleic acids and methods of the invention include, but are notlimited to, Alzheimer's Disease, Multiple Sclerosis andCreutzfeldt-Jakob disease.

Autoimmune diseases for which the invention may be used includeinflammatory muscle disease and rheumatoid arthritis.

The binding members, nucleic acids and methods of the invention may alsobe used in the treatment of cancers.

“Treatment of cancer” includes treatment of conditions caused bycancerous growth and/or vascularisation and includes the treatment ofneoplastic growths or tumours. Examples of tumours that can be treatedusing the invention are, for instance, sarcomas, including osteogenicand soft tissue sarcomas, carcinomas, e.g., breast-, lung-, bladder-,thyroid-, prostate-, colon-, rectum-, pancreas-, stomach-, liver-,uterine-, prostate cervical and ovarian carcinoma, non-small cell lungcancer, hepatocellular carcinoma, lymphomas, including Hodgkin andnon-Hodgkin lymphomas, neuroblastoma, melanoma, myeloma, Wilms tumor,and leukemias, including acute lymphoblastic leukaemia and acutemyeloblastic leukaemia, astrocytomas, gliomas and retinoblastomas.

The invention may be particularly useful in the treatment of existingcancer and in the prevention of the recurrence of cancer after initialtreatment or surgery.

The specific binding members, nucleic acids and compositions of theinvention may also be used in the treatment of other disorders mediatedby or associated with angiogenesis. Such conditions include, forexample, tumours, various autoimmune disorders, hereditary disorders,ocular disorders.

The methods of the present invention may be used to treatangiogenesis-mediated disorders including hemangioma, solid tumors,leukemia, metastasis, telangiectasia, psoriasis, scleroderma, pyogenicgranuloma, myocardial angiogenesis, Crohn's disease, plaqueneovascularization, coronary collaterals, cerebral collaterals,arteriovenous malformations, ischemic limb angiogenesis, cornealdiseases, rubeosis, neovascular glaucoma, diabetic retinopathy,retrolental fibroplasia, arthritis, diabetic neovascularization, maculardegeneration, peptic ulcer, Helicobacter related diseases, fractures,keloids, and vasculogenesis.

Specific disorders that can be treated, and compounds and compositionsfor use in the methods of the present invention, are described in moredetail below.

Ocular Disorders Mediated by Angiogenesis

Various ocular disorders are mediated by angiogenesis, and may betreated using the methods described herein. One example of a diseasemediated by angiogenesis is ocular neovascular disease, which ischaracterized by invasion of new blood vessels into the structures ofthe eye and is the most common cause of blindness. In age-relatedmacular degeneration, the associated visual problems are caused by aningrowth of choroidal capillaries through defects in Bruch's membranewith proliferation of fibrovascular tissue beneath the retinal pigmentepithelium. In the most severe form of age-related macular degeneration(known as “wet” ARMD) abnormal angiogenesis occurs under the retinaresulting in irreversible loss of vision. The loss of vision is due toscarring of the retina secondary to the bleeding from the new bloodvessels. Current treatments for “wet” ARMD utilize laser based therapyto destroy offending blood vessels. However, this treatment is not idealsince the laser can permanently scar the overlying retina and theoffending blood vessels often re-grow. An alternative treatment strategyfor macular degeneration is the use of antiangiogenesis agents toinhibit the new blood vessel formation or angiogenesis which causes themost severe visual loss from macular degeneration.

Angiogenic damage is also associated with diabetic retinopathy,retinopathy of prematurity, corneal graft rejection, neovascularglaucoma and retrolental fibroplasia. Other diseases associated withcorneal neovascularization include, but are not limited to, epidemickeratoconjunctivitis, Vitamin A deficiency, atopic keratitis, superiorlimbic keratitis, pterygium keratitis sicca, periphigoid radialkeratotomy, and corneal graph rejection. Diseases associated withretinal/choroidal neovascularization include, but are not limited to,diabetic retinopathy, macular degeneration, presumed myopia, optic pits,chronic retinal detachment, hyperviscosity syndromes, trauma andpost-laser complications. Other diseases include, but are not limitedto, diseases associated with rubeosis (neovascularization of the angle)and diseases caused by the abnormal proliferation of fibrovascular orfibrous tissue including all forms of proliferative vitreoretinopathy.

Thus, in an embodiment of the invention, the methods of the inventionmay be used in the treatment of angiogenesis-mediated ocular disorders,for example, macular degeneration.

Inflammation

The specific binding members and methods of the invention may be used inthe treatment of inflammation. By blocking the activity of CatS, thespecific binding members may prevent proper antigen presentation in‘inflammed’ cells and thus dampen the inflammatory effects.

In such an embodiment, the antibody will ideally be taken into the cellto enter the lysosome. Thus targetting methods common in the art may beused. As shown in the Examples, from the pH binding experiments, theinventors have demonstrated that the antibody will bind even at pH 4.9,suggesting that it may be effective in the lysosome.

The methods of the invention may also be used to treat angiogenesisassociated inflammation, including various forms of arthritis, such asrheumatoid arthritis and osteoarthritis.

Further, in these methods, treatment with combinations of the compoundsdescribed herein with other agents useful for treating the disorders isprovided. Such agents include, for instance, cyclooxygenase-2 (COX-2)inhibitors, which are well known to those of skill in the art.

The blood vessels in the synovial lining of the joints can undergoangiogenesis. The endothelial cells form new vascular networks andrelease factors and reactive oxygen species that lead to pannus growthand cartilage destruction. These factors are believed to activelycontribute to rheumatoid arthritis and also to osteoarthritis.Chondrocyte activation by angiogenic-related factors contributes tojoint destruction, and also promotes new bone formation. The methodsdescribed herein can be used as a therapeutic intervention to preventbone destruction and new bone formation.

Pathological angiogenesis is also believed to be involved with chronicinflammation. Examples of disorders that can be treated using themethods described herein include ulcerative colitis, Crohn's disease,bartonellosis, and atherosclerosis.

Pharmaceutical Compositions

The binding members and nucleic acids may be administered as apharmaceutical composition. Pharmaceutical compositions according to thepresent invention, and for use in accordance with the present inventionmay comprise, in addition to active ingredients, a pharmaceuticallyacceptable excipient, a carrier, buffer stabiliser or other materialswell known to those skilled in the art (see, for example, (Remington:the Science and Practice of Pharmacy, 21^(st) edition, Gennaro A R, etal, eds., Lippincott Williams & Wilkins, 2005). Such materials mayinclude buffers such as acetate, Tris, phosphate, citrate, and otherorganic acids; antioxidants; preservatives; proteins, such as serumalbumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; carbohydrates; chelatingagents; tonicifiers; and surfactants.

The pharmaceutical compositions may also contain one or more furtheractive compound selected as necessary for the particular indicationbeing treated, preferably with complementary activities that do notadversely affect the activity of the binding member, nucleic acid orcomposition of the invention. For example, in the treatment of cancer,in addition to an anti CatS specific binding member of the invention,the formulation may comprise an additional antibody which binds adifferent epitope on CatS, or an antibody to some other target such as agrowth factor that e.g. affects the growth of the particular cancer,and/or a chemotherapeutic agent.

The active ingredients (e.g. specific binding members and/orchemotherapeutic agents) may be administered via microspheres,microcapsules liposomes, other microparticulate delivery systems. Forexample, active ingredients may be entrapped within microcapsules whichmay be prepared, for example, by coacervation techniques or byinterfacial polymerization, for example, hydroxymethylcellulose orgelatin microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. For further details, see Remington:the Science and Practice of Pharmacy, 21^(st) edition, Gennaro A R, etal, eds., Lippincott Williams & Wilkins, 2005.

Sustained-release preparations may be used for delivery of activeagents. Suitable examples of sustained-release preparations includesemi-permeable matrices of solid hydrophobic polymers containing theantibody, which matrices are in the form of shaped articles, e.g. films,suppositories or microcapsules. Examples of sustained-release matricesinclude polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly (vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid andethyl-Lglutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers, and poly-D-(−)-3-hydroxybutyricacid.

As described above nucleic acids of the invention may also be used inmethods of treatment. Nucleic acid of the invention may be delivered tocells of interest using any suitable technique known in the art. Nucleicacid (optionally contained in a vector) may be delivered to a patient'scells using in vivo or ex vivo techniques. For in vivo techniques,transfection with viral vectors (such as adenovirus, Herpes simplex Ivirus, or adeno-associated virus) and lipid-based systems (useful lipidsfor lipid-mediated transfer of the gene are DOTMA, DOPE and DC-Chol, forexample) may be used (see for example, Anderson et al., Science 256:808-813 (1992). See also WO 93/25673).

In ex vivo techniques, the nucleic acid is introduced into isolatedcells of the patient with the modified cells being administered to thepatient either directly or, for example, encapsulated within porousmembranes which are implanted into the patient (see, e.g. U.S. Pat. Nos.4,892,538 and 5,283,187). Techniques available for introducing nucleicacids into viable cells may include the use of retroviral vectors,liposomes, electroporation, microinjection, cell fusion, DEAE-dextran,the calcium phosphate precipitation method, etc.

The binding member, agent, product or composition may be administered ina localised manner to a tumour site or other desired site or may bedelivered in a manner in which it targets tumour or other cells.Targeting therapies may be used to deliver the active agents morespecifically to certain types of cell, by the use of targeting systemssuch as antibody or cell specific ligands. Targeting may be desirablefor a variety of reasons, for example if the agent is unacceptablytoxic, or if it would otherwise require too high a dosage, or if itwould not otherwise be able to enter the target cells.

Dose

The binding members, nucleic acids or compositions of the invention arepreferably administered to an individual in a “therapeutically effectiveamount”, this being sufficient to show benefit to the individual. Theactual dosage regimen will depend on a number of factors including thecondition being treated, its severity, the patient being treated, theagent being used, and will be at the discretion of the physician.

The optimal dose can be determined by physicians based on a number ofparameters including, for example, age, sex, weight, severity of thecondition being treated, the active ingredient being administered andthe route of administration.

As a rough guideline, doses of antibodies may be given in amounts of 1ng/kg-500 mg/kg of patient weight.

The invention will now be described further in the followingnon-limiting examples. Reference is made to the accompanying drawings inwhich:

FIG. 1 illustrates PCR amplification of Cat S from a spleen cDNAlibrary. M: DNA 1 KB plus ladder (Invitrogen); 1: PCR productcorresponding to the mature Cat S gene specific sequence

FIG. 2 illustrates colony PCR of 6 clones (lanes 1-6) containing thegene specific region of mature Cat S protein cloned into the bacterialexpression vector pQE30. These demonstrate that the cloning has beensuccessful in all six colonies selected.

FIG. 3 illustrates scouting for optimal induction of protein expressionfrom clones 4 and 5 (from FIG. 2). Expression was induced using IPTGwhen cultures had OD values of 0.2 (early), 0.5 (mid) and 0.7 (late) OD550 nm (A-C respectively). Bacterial protein lysates were analysed bygel electrophoresis and the gel stained with Coomassie brilliant bluestain.

FIG. 4 illustrates purification of Cat S protein by immobilised metalaffinity chromatography. a) Purification profile monitoring proteinabsorbance at 260 nm, showing elution of non-specific proteins from thecolumn in the first peak, with elution of the CatS recombinant proteinseen in the broader second peak.

b) Protein elution fractions were analysed by SDS-PAGE. Lanes 1-13 arefractions eluted from the column during purification. Lane 14 is a crudebacterial lysate sample, not subjected to purification. Isolation of theCatS recombinant protein to a high purify can be clearly seen infractions 6-12

FIG. 5 illustrates analysis of test-bleeds by western blotting using anon-related recombinant protein (1) and the Cat S recombinant protein(2). Specific binding of murine sera was disclosed using goat anti-mouseHRP.

FIG. 6. ELISA screening of the hybridoma supernatants. Ag + denoteswells coated with the CatS recombinant protein whereas Ag − denotescoating with an unrelated recombinant protein as a negative control. Thehybridoma supernatants have all shown a high specificity for the CatSrecombinant protein, whilst little or no specificity for the controlprotein antigen.

FIG. 7 illustrates hybridoma supernatants analysed by western blottingagainst endogenous CatS protein from the U251 grade IV astrocytoma cellline (1) and the recombinant protein (2). Each of the unpurifiedsupernatants analysed show the ability to recognise both the inactivepro-form of CatS and the mature form from the U251 mg cell line withinwhich CatS has previously been shown to be highly expressed (ashighlighted by the boxes)

FIG. 8 Screening of hydridoma supernatants for antibodies which caninhibit CatS-mediated hydrolysis of the fluorogenic substrateCbz-Val-Val-Arg-AMC. Asterisk labelled columns represent the hydridomaclones taken forward for further examination. Some of the mostinhibitory clones were not able to be propagated further and thereforewere not chosen. The double asterisked clone represents the finalselected inhibitory antibody

FIG. 9 illustrates representative elution profile of CatS specificmonoclonal antibodies. The lower trace represents absorbance (260 nm)with purification of the monoclonal antibody. The peak on the rightindicates the elution of the monoclonal antibody and the bars belowrepresent which fractions contain the eluted antibody.

FIG. 10 illustrates results from the isotyping of the monoclonalantibodies. Values greater than 0.8 and highlighted in bold arepositive. Four of the antibodies tested were IgG1 antibodies with twobeing IgM and all six antibodies have kappa light chains. Ab 1, labelledwith the asterisk represents the antibody finally identified as aninhibitory antibody. Antibody 2 is CatS specific, but non-inhibitory,and is used in the later invasion assays as the isotype control.

FIG. 11 illustrates fluorometric assays demonstrating inhibition of CatS activity in the presence of varying concentrations of two purifiedCatS MAb antibodies. MAb1 shows dose-dependent inhibition ofCbz-Val-Val-Arg-AMC cleavage by CatS. MAb2 in this panel does not haveany effect on proteolytic activity of CatS.

FIG. 12. Quantification of inhibitory activity of CatS MAb1 (Mab 1e11).Rates from the progress curves of (Mab 1E11) [FIG. 11], which wereindicative of a slow binding inhibitor, were fitted by non-linearregression using GraFit software according to the method of Morrison andWalsh, 1988. Using this approach an inhibition constant (K_(i)) of 533nM was calculated

FIG. 13. MAb1 does not inhibit CatK. Progression curves plotted showinghydrolysis of Cbz-Phe-Arg-AMC by purified CatK in the presence of theCatS inhibitory mAb.

FIG. 14 MAb1 does not inhibit CatL. Progression curves plotted showinghydrolysis of Cbz-Phe-Arg-AMC by purified CatL in the presence of theCatS inhibitory mAb.

FIG. 15 MAb1 specifically binds CatS on western blot. The antibody wasused to probe against human cathepsin B, L, S and K (100 ng each).

FIG. 16 CatS Mab1 binds specifically to CatS by ELISA. Graphdemonstrates results of antigen immobilised ELISA performed with CatS,B, L and K recombinant proteins (100 ng per well) to which the CatSinhibitory antibody was incubated in a range of dilutions, from 1×10⁻⁷to 1×10⁻¹² M.

FIG. 17 CatS MAb1 binds to CatS over pH range. Graph shows results of animmobilised antigen ELISA showing the ability of differentconcentrations of the CatS inhibitory antibody to maintain its affinityfor CatS over a wide pH range; from neutral pH 7.5 to pH 4.9.

FIG. 18 illustrates the RT-PCR amplification of variable region of theheavy chain (HV) and the variable region of the light chain (LV) and VLregions of the inhibitory monoclonal antibody. The RT-PCR was performedfrom mRNA isolated from the hydridoma expressing and secreting CatSMAb1.

FIG. 19 illustrates the results from the colony PCR on the TOPO cloningof (a) VH and (b) VL. All 16 colonies analysed for the VH region appearpositive by colony PCR, and all but two are positive for the VL region.

FIG. 20 illustrates the consensus amino acid sequence of the VH and VLregions of the inhibitory monoclonal antibody with CDR's highlighted inbold and underlined, as determined from DNA sequencing of the VH and VLregions.

FIG. 21 RT-PCR of CatS to identify expression in a range of tumour celllines, grade IV astrocytoma (U251), prostate (PC3), colorectal (HCT116)and breast (MCF7).

FIG. 22 shows the effect of the CatS inhibitory monoclonal antibody inan in vitro invasion assay on the PC3 prostate carcinoma cell line. Theinvasion assay was performed in the presence of increasingconcentrations of the inhibitory antibody (0-200 nM), showing areduction in tumour cell invasion of up to 39%. A non-related isotypecontrol mAb had no effect on invasion, while another CatS mAb (which wehave previously shown could bind to but not inhibit the proteolyticactivity of CatS) had no significant effect on invasion at 200 nM.Representative photomicrographs are also shown.

FIG. 23 illustrates the effect of the CatS inhibitory monoclonalantibody in an in vitro invasion assay on the U251 mg astrocytoma cellline. The invasion assay was performed in the presence of increasingconcentrations of the inhibitory antibody (0-200 nM), showing areduction in tumour cell invasion of up to 29%. A non-related isotypecontrol mAb and a non-inhibitory CatS MAb had no significant effect oninvasion. Representative photomicrographs are also shown.

FIG. 24 illustrates the effect of the CatS inhibitory monoclonalantibody in an in vitro invasion assay on the HCT116 colorectalcarcinoma cell line. The invasion assay was performed in the presence ofincreasing concentrations of the inhibitory antibody (0-200 nM), showinga reduction in tumour cell invasion of up to 64%. A non-related isotypecontrol mAb and the non-inhibitory CatS MAb had no significant effect oninvasion. Representative photomicrographs are also shown

FIG. 25 illustrates the effect of the CatS inhibitory monoclonalantibody in an in vitro invasion assay on the MDA-MB-231 breastcarcinoma cell line. The invasion assay was performed in the presence ofincreasing concentrations of the inhibitory antibody (0-200 n), showinga reduction in tumour cell invasion of up to 32%. A non-related isotypecontrol mAb and the non-inhibitory CatS MAb had no significant effect oninvasion. Representative photomicrographs are also shown.

FIG. 26 illustrates the effect of the CatS inhibitory monoclonalantibody in an in vitro invasion assay on the MCF7 breast carcinoma cellline. The invasion assay was performed in the presence of 200 nM of acontrol isotype monoclonal antibody and 200 nm of the inhibitoryantibody, showing a reduction in tumour cell invasion of 63%.Representative photomicrographs are also shown.

FIG. 27 illustrates the results of a Matrigel assay, illustrating thatcapillary tubule formation is inhibited in the presence Mab 1E11.

Materials and Methods.

Cloning

The DNA sequence encoding the mature Cat S protein was amplified by PCRfrom a spleen cDNA library using gene-specific primers encoding BamH1and Sal1 restriction sites (denoted by lower case letters) (FIG. 1).Sense: TTT TTT gga tcc TTG CCT GAT TCT GTG GAC TGG AGA Antisense: TTTTTT gtc gac CTA GAT TTC TGG GTA AGA GG

The Cat S gene was cloned into the bacterial expression vector pQE30allowing the incorporation of a hexahistidine tag onto the N-terminus ofthe recombinant protein. This construct was then used to transformcompetent TOP10F′ E. coli cells (Invitrogen). Positive transformantswere selected by colony PCR using vector-specific primers flanking themultiple cloning site (FIG. 2).

Expression of Recombinant Cats Protein

The positive clones were propagated overnight at 37° C. in 5 mls ofLuria-Bertani (LB) broth supplemented with 50 μM ampicillin. A 300 μlaliquot of this culture was retained for inoculation of secondarycultures and the remainder of the sample was miniprepped using theQiagen miniprep kit and the sequence verified by DNA sequencing.

Three secondary cultures were inoculated to allow visualisation ofprotein expression. The cultures were induced with IPTG (finalconcentration 1 mM) when the cultures had an OD of 0.2, 0.5 and 1.0(A₅₅₀) respectively and then left for 4 hrs at 37° C. The cells werethen harvested by centrifugation at 4000 rpm for 15 mins and the pelletresuspended in 1 ml of PBS/0.1% Igepal supplemented with 1 μl oflysonase. Samples were then analysed by SDS-PAGE and western blotting toconfirm expression of the protein. The SDS-PAGE gel was stainedovernight in coomassie blue and destained the following day (FIG. 3).

The recombinant Cat S protein was then expressed in 500 mls of LB brothsupplemented with ampicillin, using the secondary culture as aninoculant and induced with IPTG once the culture had reached the optimaloptical density. The culture was centrifuged at 5000 rpm for 15 mins andthe pellet retained for protein purification.

Protein Purification

The induced recombinant protein was solubilised in 50 mls of 8 M ureabuffer (480 g Urea, 29 g NaCl, 3.12 g NaH2PO4 (dihydrate), 0.34 gImidazole) overnight. The solution was centrifuged at 6000 rpm for 1 hr,after which the supernatant was filtered using 0.8 μm gyrodisc filtersbefore purification.

The protein was purified by its N-terminal hexahistidine tag andrefolded using on-column refolding by immobilized metal affinitychromatography. Chelating hi-trap columns (Amersham Biosciences) werecharged using 100 mM nickel sulphate before attachment to the Aktaprime.Refolding takes place by the exchange of the 8 M urea buffer with a 5 mMimidazole wash buffer (29 g NaCl, 3.12 g NaH2PO4 (dihydrate) 0.34 gImidazole, pH 8.0) and elution of the protein using a 500 mM imidazoleelution buffer (29 g NaCl, 3.12 g NaH2PO4 (dihydrate), 34 g Imidazole).The elution profile of the purified recombinant protein was recorded andcan be seen in FIG. 4 a.

The eluted fractions were subjected to SDS-PAGE analysis to confirmrecombinant protein presence in eluted fractions. The gels were stainedwith coomassie blue overnight and subsequently destained to determinethe fractions containing the Cat S protein (FIG. 4 b).

Antibody Generation

The refolded protein was used as an immunogen to generate monoclonalantibodies. Five BALB/C mice were immunized at three weekly intervalswith 150 μg of purified recombinant protein and the antibody titre wasanalysed after boosts three and five. A test bleed was taken from eachanimal and tested at 1:1000 dilutions in western blotting against 100 ngof antigen. Blots were developed using 3,3′-diaminobenzidine (DAB) (asdescribed earlier) (FIG. 5).

After the fifth boost, the spleen was removed from the mouse and theantibody producing B cells were fused with SP2 myeloma cells followingstandard protocols. Five days after the hybridoma fusion, the HAT mediawas refreshed and after a further five days, the plates were examinedfor cell growth. Clones were screened by ELISA against recombinantprotein and selected positive hybridomas were cloned twice by limitingdilution.

ELISA

The monoclonal antibodies were screened by ELISA to determine whichclones should be expanded. Maxi Sorb 96 well plates were coated withrecombinant antigen by adding 100 μl of coating buffer (Buffer A: 0.42 gsodium bicarbonate/100 μl H₂O, Buffer B: 0.53 g sodium carbonate/100 μlH₂O, pH 9.5) containing the screening antigen to each well (100ng/well). A control antigen was also used to eliminate non-specificclones. The plates were incubated at 37° C. for 1 hr to allow theantigen to bind to the well and then blocked for 1 hr at roomtemperature by adding 200 μl PBS/3% BSA to each well.

The blocking solution was removed from the plates and 100 μl ofhybridoma supernatant was added to a positive antigen and a controlantigen well. The screening plates were incubated with supernatant for 1hr on a rocker at room temperature. The plates were washed three timeswith PBS-T, after which 100 μl of goat anti-mouse HRP conjugatedsecondary antibody (1:3000) was added to each well and incubated for 1hr at room temperature. The plates were washed three times with PBS-Tand 100 μl of 3,3′,5,5′-tetramethylbenzidine (TMB) was added to eachwell and incubated for 5 mins at 37° C. Positive wells were indicated bya colour development and the reaction was stopped by addition of 50 g μlM HCL. Plates were read by a spectrophotometer at 450 nm and samplesdisplaying a positive reading in the screening well (+) with a negativereading in the control well (−) were chosen for further work (FIG. 6).The cells from the original wells were transferred into a 24 well plateand grown up.

Western Blotting

The supernatants from the hybridoma cell lines were analysed by westernblotting to determine the ability of the monoclonal antibodies to detectendogenous native Cat S protein in the U251 mg grade IV astrocytoma cellline, in which Cat S is highly expressed. A 30 μg/ml aliquot of U251 mgwhole cell lysate was separated by SDS-PAGE and transferred ontoHybond-C Extra nitrocellulose membrane (Amersham Biosciences). Themembrane was blocked by incubation in PBS/5% marvel for 1 hr at roomtemperature, after which it was rinsed briefly in PBS. The monoclonalantibodies were used at a 1:500 dilution in PBS and incubated on themembrane overnight at 4° C. while gently rocking. The blot was thenrinsed three times with PBS/1% marvel and 0.1% Tween-20 and thenincubated with the goat anti-mouse HRP conjugated secondary antibody ata 1:3000 dilution for 1 hr at room temperature while shaking. The blotwas then rinsed three times with the PBS/1% marvel and 0.1% Tween-20solution, followed by a short rinse in PBS. The blot was incubated withECL plus substrate (Amersham Bioscicences) for 5 mins at roomtemperature before development using Kodak photographic film under safelight conditions (FIG. 7).

High Throughput Screening to Identify Potential Inhibitory Antibodies

The inhibitory effect of the monoclonal antibodies were determined by afluorometric assay, using a Cat S synthetic substrate Z-Val-Val-Arg-AMC(Bachem) and recombinant human Cat S (Calbiochem). The recombinant Cat Senzyme was activated at 37° C. in sodium acetate buffer (100 mM sodiumacetate, 1 mM EDTA, 0.1% Brij90, pH 5.5), and 2 mM dithiothreitol (DTT)for 30 mins prior to assay. Assays were carried out using 190 μl ofsodium acetate/DTT buffer (90:1), 1 μl of activated Cat S, 12.5 μl of 10mM Cbz-val-Val-Arg-AMC substrate and 50 μl of non-purified monoclonalantibody supernatant. Fluorescence was measured at 375 nm excitation and460 nm emission wavelengths every 5 mins for 4 hrs (FIG. 8). From thisfive monoclonal antibody secreting cell lines were selected forlarge-scale growth, purification. The hybridoma supernatant was purifiedby affinity chromatography using either an protein G or protein M column(dependent on isotyping results) (representative FIG. 9).

Isotyping of Monoclonal Antibodies

The monoclonal antibodies selected for large scale growth were isotypedprior to purification using the ImmunoPure® monoclonal antibodyisotyping kit from Pierce. Ten wells on a 96-well plate were coated with50 μl of a goat anti-mouse coating antibody and incubated at roomtemperature for 2 hrs. The wells were blocked to prevent non-specificbinding, using 125 μl of blocking solution and incubated at roomtemperature for 1 hr. The wells were then washed four times with 125 μlof wash buffer. Nine of the ten wells were incubated with 50 μl ofhybridoma supernatant, with 50 μl of positive control solution added tothe tenth well and incubated at room temperature for 1 hr. The wellswere washed once again using 125 μl of wash buffer before addition of 50μl of subclass-specific anti-mouse immunoglobulins and controls to theten separate wells and incubated at room temperature for 1 hr. The wellswere washed four times with 125 μl of wash buffer, after which 100 μl ofABTS substrate solution was added to each well and allowed to react atroom temperature for 30 mins. The results were read using aspectrophotometer at 405 nm (FIG. 10).

Fluorometric Assay

The purified monoclonal antibodies were analysed by fluorometric assayfor quantification of their ability to inhibit the activity of Cat S.The principles of this assay are the same as that previously used.Fluorescence was measured at 375 nm excitation and 460 nm emissionwavelengths every minute for 1 hr. (FIG. 11). These progress curves areindicative of the action of a slow-binding reversible inhibitor. Theapparent first order rate order curves produced were then subjected tonon-linear regression analysis (Morrison and Walsh, 1988) using GraFitsoftware as shown (FIG. 12), producing a K_(i) of 533 nM. Controlfluorometric assays using cathepsins L and K were performed as describedabove using 50 μM of the fluorogenic substrate Cbz-Phe-Arg-AMC (FIGS. 14and 15), demonstrating that the inhibitory activity towards CatS wasindeed specific.

Specificity of Antibody Binding

Western blots were performed as described before to show specificity ofthe antibody binding to CatS and not to CatB, L or K. Briefly, 100 ng ofrecombinant CatS, K, L and B were loaded onto a SDS-PAGE gel, thentransferred onto Hybond-C Extra nitrocellulose membrane (AmershamBiosciences). The membrane was blocked by incubation in PBS/5% marvelfor 1 hr at room temperature, after which it was rinsed briefly in PBS.The monoclonal antibodies were used at a 1:500 dilution in PBS andincubated on the membrane overnight at 4° C. while gently rocking. Theblot was then rinsed three times with PBS/1% marvel and 0.1% Tween-20and then incubated with the goat anti-mouse HRP conjugated secondaryantibody at a 1:3000 dilution for 1 hr at room temperature whileshaking. The blot was then rinsed three times with the PBS/1% marvel and0.1% Tween-20 solution, followed by a short rinse in PBS. The blot wasincubated with ECL plus substrate (Amersham Biosciences) for 5 mins atroom temperature before development using Kodak photographic film undersafe light conditions (FIG. 15).

Specificity of binding was also determined by Antigen-immobilisedELISAs. These were performed as described earlier to determinespecificity and affinity of the antibody for CatS. To determinespecificity, 96-well plates were coated with 100 ng/well of cathepsinsS, L, K and B in duplicate and incubated with predeterminedconcentrations of the CatS mAb (1×10⁻⁷ to 1×10¹⁻²) (FIG. 16).

In addition the ability of the antibody to bind to CatS was alsoexamined in a pH range. This ELISA was performed on a 96-well platescoated with 100 ng/well of CatS antigen and incubated with predeterminedconcentrations of the CatS mAb (1×10⁻⁷ to 1×10⁻¹²) in a series ofdifferent pH points applying the antibody dilutions to the plate in 50mM Bis-Tris.HCl buffer adjusted to the given pH points, allowingantibody affinity to be determined (FIG. 17). From this it was clearthat binding was similar at pH 5.5 to 7.5, but slightly reduced at pH4.9.

Monoclonal Sequencing

For the sequencing of the inhibitory monoclonal antibody, the RNA wasextracted from the hybridoma cell line and the VH and VL regionsamplified by RT-PCR (FIG. 18). The PCR products were cloned using theTOPO TA kit from Invitrogen and positive colonies were verified bycolony PCR using vector specific primers (FIG. 19) and DNA sequencing toderived the consensus sequence of the VH and VL regions of theinhibitory monoclonal antibody (FIG. 20).

Confirmation of the Presence of CatS in Tumour Cell Lines by RT-PCR

RNA was extracted from U251 mg, MCF7, HCT116 and PC3 cell lines usingthe Absolutely RNA™ RT-PCR Miniprep kit (Stratagene) according tomanufacturer's instructions and quantified using a spectrophotometer.RT-PCR was performed using the One-Step RT-PCR kit (Qiagen) under thefollowing conditions: 50° C. for 30 min, 95° C. for 15 min, and 40cycles of 94° C. for 1 min, 55° C. for 1 min 30 sec and 72° C. for 1min, followed finally by a 72° C. for 10 min. Primer sequences for CatSwere as follows; CatS F: GGGTACCTCATGTGACAAG, CatS R:TCACTTCTTCACTGGTCATG; Amplification of the β-actin gene was used as aninternal control to demonstrate equal loading; Actin F:ATCTGGCACCACACCTTCTACAATGAGCTGCG Actin R:CGTCATACTCCTGCTTGCTGATCCACATCTGC. RT-PCR products were analysed byagarose gel electrophoresis (FIG. 21).

In-Vitro Invasion Assays

In-vitro invasion assays were performed using a modified Boyden chamberwith 12-μm pore membranes (Costar Transwell plates, Corning CostarCorp., Cambridge, Mass., USA). The membranes were coated with Matrigel(100 μg/cm²) (Becton Dickinson, Oxford, UK) and allowed to dry overnightin a laminar flow hood. Cells were added to each well in 500 μl ofserum-free medium in the presence of predetermined concentrations of theCatS inhibitory antibody or control antibody. All assays were carriedout in triplicate and invasion plates were incubated at 37° C. and 5%CO₂ for 24 hours after which cells remaining on the upper surface of themembrane were removed and invaded cells fixed in Carnoy's fixative for15 minutes. After drying, the nuclei of the invaded cells were stainedwith Hoechst 33258 (50 ng/ml) in PBS for 30 minutes at room temperature.The chamber insert was washed twice in PBS, mounted in Citifluor andinvaded cells were viewed with a Nikon Eclipse TE300 fluorescentmicroscope. Ten digital images of representative fields from each of thetriplicate membranes were taken using a Nikon DXM1200 digital camera atmagnification of ×20. The results were analysed using Lucia GF 4.60 byLaboratory Imaging and were expressed as a percentage of invaded cells(FIGS. 22-26).

Capillary-Like Tube Formation Assay

The anti-angiogenic properties of the CatS mAb was assessed using aHUVEC cells microtubule formation assay. The effect of the antibody onendothelial cell tube formation was assessed as follows: Two hundredmicroliter of Matrigel (10 mg/ml) was applied to pre-cooled 48-wellplates, incubated for 10 min at 4° C. and then allowed to polymerize for1 h at 37° C. Cells were suspended in endothelial growth cell medium MV(Promocell), containing 200 nM of the appropriate antibody. Five hundredmicroliter (1×10⁵ cells) was added to each well. As controls, cells wereincubated with vehicle-only control medium containing the appropriatevolumes of PBS. After 24 h incubation at 37° C. and 5% CO2, cells wereviewed using a Nikon Eclipse TE300 microscope.

Cells grown in the presence of the 1E11 Mab displayed an inability toform microtubules indicating that CatS specific antibodies have theability to disrupt microtubule formation, indicating anti-tumourogenicproperties influencing cell migration and angiogenisis (FIG. 27). Cellsgrown in the presence of an isotype control antibody displayed normalmicrotubule formation

All documents referred to in this specification are herein incorporatedby reference. Various modifications and variations to the describedembodiments of the inventions will be apparent to those skilled in theart without departing from the scope and spirit of the invention.Although the invention has been described in connection with specificpreferred embodiments, it should be understood that the invention asclaimed should not be unduly limited to such specific embodiments.Indeed, various modifications of the described modes of carrying out theinvention which are obvious to those skilled in the art are intended tobe covered by the present invention.

REFERENCES

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1. A method of treating a condition associated with angiogenesis in apatient in need of treatment thereof, said method comprisingadministration of an antibody or fragment thereof or a nucleic acidencoding said antibody or thereof to said patient wherein the antibodyor fragment thereof binds cathepsin S and inhibits its proteolyticactivity.
 2. The method according to claim 1, wherein the conditionassociated with angiogenesis is cancer, an angiogenesis associatedinflammatory condition or an ocular disease.
 3. The method according toclaim 2, wherein the condition is sarcoma, breast cancer, lung cancer,bladder cancer, thyroid cancer, prostate cancer, colon cancer, rectumcancer, pancreas cancer, stomach cancer, liver cancer, uterine cancer,prostate, cervical and ovarian carcinoma, non-small cell lung cancer,hepatocellular carcinoma, lymphoma, neuroblastoma, melanoma, myeloma,Wilms tumor, leukemia, astrocytoma, glioma or retinoblastoma.
 4. Themethod according to claim 2, wherein the condition is maculardegeneration.
 5. The method according to claim 2, the condition isbreast cancer.
 6. The method according to claim 2, wherein the conditionis prostate cancer.
 7. The method according to claim 2, wherein thecondition is colorectal cancer.
 8. The method according to claim 2,wherein the condition is astrocytomas.
 9. The method according to claim2, wherein the condition is rheumatoid arthritis or osteoarthritis. 10.The method according to claim 2, wherein the condition is ulcerativecolitis, Crohn's disease, bartonellosis, or atherosclerosis. 11-20.(canceled)
 21. A specific binding member which binds cathepsin S andinhibits its proteolytic activity wherein the specific binding membercomprises an antigen binding domain comprising (i) a CDR with an aminoacid sequence Seq ID No: 1 a CDR with an amino acid sequence Seq ID No:2, and a CDR with amino acid sequence Seq ID No: 3, and/or (ii) a CDRwith an amino acid sequence Seq ID No.: 4, a CDR with an amino acidsequence Seq ID No: 5, and a CDR with an amino acid sequence Seq ID No:6.
 22. The specific binding member according to claim 21 wherein theantigen binding domain comprises (i) a CDR with an amino acid sequenceSeq ID No: 1, a CDR with an amino acid sequence Seq ID No: 2, and a CDRwith an amino acid sequence Seq ID No: 3; and (ii) a CDR with an aminoacid sequence Seq D) No: 4, a CDR with an amino acid sequence Seq ID No:5, and a CDR with an amino acid sequence Seq ID No:
 6. 23. The specificbinding member according to claim 21, wherein the antibody V_(H) domaincomprises CDRs with amino acid sequences Seq ID No: 1, Seq ID No: 2 andSeq ID No: 3 as CDRs 1, 2 and 3 respectively.
 24. The specific bindingmember according to claim 23 wherein the antibody V_(H) domain comprisesthe amino acid sequence Seq ID No:
 7. 25. The specific binding memberaccording to claim 21, wherein the antibody V_(L) domain comprises CDRswith amino acid sequences Seq ID No: 4, Seq ID No: 5 and Seq ID No: 6 asCDRs 1, 2 and 3 respectively.
 26. The specific binding member accordingto claim 27 wherein the antibody V_(L) domain comprises the amino acidsequence Seq ID No:
 8. 27. The specific binding member according toclaim 21, wherein the specific binding member inhibits the proteolyticactivity of cathepsin S with a potency at least 25% of that of anantibody comprising an antibody V_(H) domain having the amino acidsequence Seq ID No: 7 and an antibody V_(L) domain having the amino acidsequence Seq ID No:
 8. 28. The specific binding member according toclaim 21, wherein the specific binding member is a whole antibody. 29.The specific binding member according to claim 21, wherein the specificbinding member comprises an scFv antibody molecule.
 30. A nucleic acidencoding a specific binding member according to claim
 21. 31. Apharmaceutical composition comprising a specific binding memberaccording to claim 21 or a nucleic acid according to claim
 30. 32. Aspecific binding member according to claim
 21. 33. A method of producinga specific binding member capable of binding cathepsin S, said methodcomprising expressing the nucleic acid according to claim 30 in a hostcell and isolating said specific binding member from said cell. 34-42.(canceled)